High Rates of Potentially Infectious Tuberculosis and Multidrug-Resistant Tuberculosis (MDR-TB) among Hospital Inpatients in KwaZulu Natal, South Africa Indicate Risk of Nosocomial Transmission

Background: Nosocomial transmission has been implicated as a key factor in the outbreak of extensively drug resistant (XDR) and multidrug-resistant (MDR-TB) tuberculosis at Church of Scotland Hospital (CoSH), in KwaZulu-Natal (KZN), South Africa. The aim of this study was to quantify the burden of potentially infectious tuberculosis and the proportion of drug resistance among hospital inpatients throughout the province of KZN. Methods: Inpatients with current cough, capable of producing sputum were selected from 19 public hospitals in KZN. After informed consent, demographic and clinical data, and sputum samples were collected. Samples were processed for fluorescent microscopy, liquid culture and first and second-line anti-tuberculosis drug susceptibility testing. Results: There were a total of 2,964 inpatients where sampling was done. About 1,585 inpatients (53%) had a current cough and sufficient microbiological and clinical data for inclusion. Mycobacterium tuberculosis was isolated from 543 inpatients (34% of those tested and 18% of all inpatients). Eighty-four (15%) inpatients with TB were found to be MDR-TB infected and 16 (3%) had XDR-TB. There was no association between the prevalence of MDR-TB and proximity to CoSH. Among patients with microbiologically confirmed TB, MDR/XDR-TB was associated with male sex, a longer length of stay between hospital admission and date of sample collection, and current or previous TB treatment. Conclusions: One in five inpatients had potentially infectious TB. This is an underestimate since patients without current cough were not tested. MDR-TB was frequently observed and was found in nearly one in six active TB inpatients. While present at lower levels than the original outbreak report at CoSH, XDR-TB was detected in hospitals throughout KZN. The high burden of potentially infectious TB and confirmed MDR-TB, much of it undiagnosed, indicates a serious risk for nosocomial transmission and the need for intensified infection control within the inpatient setting

N-Acetyltransferase 2 Genotypes among Zulu-Speaking South Africans and Isoniazid and N-Acetyl-Isoniazid Pharmacokinetics during Antituberculosis Treatment.

The distribution of N-acetyltransferase 2 gene (NAT2) polymorphisms varies considerably among different ethnic groups. Information on NAT2 single-nucleotide polymorphisms in the South African population is limited. We investigated NAT2 polymorphisms and their effect on isoniazid pharmacokinetics (PK) in Zulu black HIV-infected South Africans in Durban, South Africa. HIV-infected participants with culture-confirmed pulmonary tuberculosis (TB) were enrolled from two unrelated studies. Participants with culture-confirmed pulmonary TB were genotyped for the NAT2 polymorphisms 282C>T, 341T>C, 481C>T, 857G>A, 590G>A, and 803A>G using Life Technologies prevalidated TaqMan assays (Life Technologies, Paisley, UK). Participants underwent sampling for determination of plasma isoniazid and N-acetyl-isoniazid concentrations. Among the 120 patients, 63/120 (52.5%) were slow metabolizers (NAT2*5/*5), 43/120 (35.8%) had an intermediate metabolism genotype (NAT2*5/12), and 12/120 (11.7%) had a rapid metabolism genotype (NAT2*4/*11, NAT2*11/12, and NAT2*12/12). The NAT2 alleles evaluated in this study were *4, *5C, *5D, *5E, *5J, *5K, *5KA, *5T, *11A, *12A/12C, and *12M. NAT2*5 was the most frequent allele (70.4%), followed by NAT2*12 (27.9%). Fifty-eight of 60 participants in study 1 had PK results. The median area under the concentration-time curve from 0 to infinity (AUC0-∞) was 5.53 (interquartile range [IQR], 3.63 to 9.12 μg h/ml), and the maximum concentration (Cmax) was 1.47 μg/ml (IQR, 1.14 to 1.89 μg/ml). Thirty-four of 40 participants in study 2 had both PK results and NAT2 genotyping results. The median AUC0-∞ was 10.76 μg·h/ml (IQR, 8.24 to 28.96 μg·h/ml), and the Cmax was 3.14 μg/ml (IQR, 2.39 to 4.34 μg/ml). Individual polymorphisms were not equally distributed, with some being represented in small numbers. The genotype did not correlate with the phenotype, with those with a rapid acetylator genotype showing higher AUC0-∞ values than those with a slow acetylator genotype, but the difference was not significant (P = 0.43). There was a high prevalence of slow acetylator genotypes, followed by intermediate and then rapid acetylator genotypes. The poor concordance between genotype and phenotype suggests that other factors or genetic loci influence isoniazid metabolism, and these warrant further investigation in this population

Population-level emergence of bedaquiline and clofazimine resistance-associated variants among patients with drug-resistant tuberculosis in southern Africa: a phenotypic and phylogenetic analysis.

BACKGROUND: Bedaquiline and clofazimine are important drugs in the treatment of drug-resistant tuberculosis and are commonly used across southern Africa, although drug susceptibility testing is not routinely performed. In this study, we did a genotypic and phenotypic analysis of drug-resistant Mycobacterium tuberculosis isolates from cohort studies in hospitals in KwaZulu-Natal, South Africa, to identify resistance-associated variants (RAVs) and assess the extent of clofazimine and bedaquiline cross-resistance. We also used a comprehensive dataset of whole-genome sequences to investigate the phylogenetic and geographical distribution of bedaquiline and clofazimine RAVs in southern Africa. METHODS: In this study, we included M tuberculosis isolates reported from the PRAXIS study of patients with drug-resistant tuberculosis treated with bedaquiline (King Dinuzulu Hospital, Durban) and three other cohort studies of drug-resistant tuberculosis in other KwaZulu-Natal hospitals, and sequential isolates from six persistently culture-positive patients with extensively drug-resistant tuberculosis at the KwaZulu-Natal provincial referral laboratory. Samples were collected between 2013 and 2019. Microbiological cultures were done as part of all parent studies. We sequenced whole genomes of included isolates and measured bedaquiline and clofazimine minimum inhibitory concentrations (MICs) for isolates identified as carrying any Rv0678 variant or previously published atpE, pepQ, and Rv1979c RAVs, which were the subject of the phenotypic study. We combined all whole-genome sequences of M tuberculosis obtained in this study with publicly available sequence data from other tuberculosis studies in southern Africa (defined as the countries of the Southern African Development Community), including isolates with Rv0678 variants identified by screening public genomic databases. We used this extended dataset to reconstruct phylogenetic relationships across lineage 2 and 4 M tuberculosis isolates. FINDINGS: We sequenced the whole genome of 648 isolates from 385 patients with drug-resistant tuberculosis recruited into cohort studies in KwaZulu-Natal, and 28 isolates from six patients from the KwaZulu-Natal referral laboratory. We identified 30 isolates with Rv0678 RAVs from 16 (4%) of 391 patients. We did not identify any atpE, pepQ, or Rv1979c RAVs. MICs were measured for 21 isolates with Rv0678 RAVs. MICs were above the critical concentration for bedaquiline resistance in nine (43%) of 21 isolates, in the intermediate category in nine (43%) isolates, and within the wild-type range in three (14%) isolates. Clofazimine MICs in genetically wild-type isolates ranged from 0·12-0·5 μg/mL, and in isolates with RAVs from 0·25-4·0 μg/mL. Phylogenetic analysis of the extended dataset including M tuberculosis isolates from southern Africa resolved multiple emergences of Rv0678 variants in lineages 2 and 4, documented two likely nosocomial transmission events, and identified the spread of a possibly bedaquiline and clofazimine cross-resistant clone in eSwatini. We also identified four patients with pepQ frameshift mutations that may confer resistance. INTERPRETATION: Bedaquiline and clofazimine cross-resistance in southern Africa is emerging repeatedly, with evidence of onward transmission largely due to Rv0678 mutations in M tuberculosis. Roll-out of bedaquiline and clofazimine treatment in the setting of limited drug susceptibility testing could allow further spread of resistance. Designing strong regimens would help reduce the emergence of resistance. Drug susceptibility testing is required to identify where resistance does emerge. FUNDING: Wellcome Trust, National Institute of Allergy and Infectious Diseases and National Center for Advancing Translational Sciences of the National Institutes of Health

Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture.

BACKGROUND: Repeated culture reduces within-sample Mycobacterium tuberculosis genetic diversity due to selection of clones suited to growth in culture and/or random loss of lineages, but it is not known to what extent omitting the culture step altogether alters genetic diversity. We compared M. tuberculosis whole genome sequences generated from 33 paired clinical samples using two methods. In one method DNA was extracted directly from sputum then enriched with custom-designed SureSelect (Agilent) oligonucleotide baits and in the other it was extracted from mycobacterial growth indicator tube (MGIT) culture. RESULTS: DNA directly sequenced from sputum showed significantly more within-sample diversity than that from MGIT culture (median 5.0 vs 4.5 heterozygous alleles per sample, p = 0.04). Resistance associated variants present as HAs occurred in four patients, and in two cases may provide a genotypic explanation for phenotypic resistance. CONCLUSIONS: Culture-free M. tuberculosis whole genome sequencing detects more within-sample diversity than a leading culture-based method and may allow detection of mycobacteria that are not actively replicating

Correction to: Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture.

He authors reported that one of the authors' names was typeset incorrectly in the authorship list

Evaluation of a synthetic peptide for the detection of anti-Mycobacterium tuberculosis curli pili IgG antibodies in patients with pulmonary tuberculosis.

CAPRISA, 2018.Abstract available in pdf

Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells

A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. DOI: http://dx.doi.org/10.7554/eLife.22028.00

Systematic Genetic Nomenclature for Type VII Secretion Systems

CITATION: Bitter, W., et al. 2009. Systematic genetic nomenclature for type VII secretion systems. PLoS Pathogens, 5(10): 1-6, doi: 10.1371/journal.ppat.1000507.The original publication is available at http://journals.plos.org/plospathogensMycobacteria, such as the etiological agent of human tuberculosis, Mycobacterium tuberculosis, are protected by an impermeable cell envelope composed of an inner cytoplasmic membrane, a peptidoglycan layer, an arabinogalactan layer, and an outer membrane. This second membrane consists of covalently linked, tightly packed long-chain mycolic acids [1,2] and noncovalently bound shorter lipids involved in pathogenicity [3–5]. To ensure protein transport across this complex cell envelope, mycobacteria use various secretion pathways, such as the SecA1-mediated general secretory pathway [6,7], an alternative SecA2-operated pathway [8], a twin-arginine translocation system [9,10], and a specialized secretion pathway variously named ESAT-6-, SNM-, ESX-, or type VII secretion [11–16]. The latter pathway, hereafter referred to as type VII secretion (T7S), has recently become a large and competitive research topic that is closely linked to studies of host–pathogen interactions of M. tuberculosis [17] and other pathogenic mycobacteria [16]. Molecular details are just beginning to be revealed [18–22] showing that T7S systems are complex machineries with multiple components and multiple substrates. Despite their biological importance, there has been a lack of a clear naming policy for the components and substrates of these systems. As there are multiple paralogous T7S systems within the Mycobacteria and orthologous systems in related bacteria, we are concerned that, without a unified nomenclature system, a multitude of redundant and obscure gene names will be used that will inevitably lead to confusion and hinder future progress. In this opinion piece we will therefore propose and introduce a systematic nomenclature with guidelines for name selection of new components that will greatly facilitate communication and understanding in this rapidly developing field of research.http://journals.plos.org/plospathogens/article?id=10.1371%2Fjournal.ppat.1000507Publisher's versio